title: Quaternary structure of SecA in solution and bound to SecYEG probed at the single molecule level.
abstract: Dual-color fluorescence-burst analysis (DCFBA) was applied to measure the quaternary structure and high-affinity binding of the bacterial motor protein SecA to the protein-conducting channel SecYEG reconstituted into lipid vesicles. DCFBA is an equilibrium technique that enables the direct observation and quantification of protein-protein interactions at the single molecule level. SecA binds to SecYEG as a dimer with a nucleotide- and preprotein-dependent dissociation constant. One of the SecA protomers binds SecYEG in a salt-resistant manner, whereas binding of the second protomer is salt sensitive. Because protein translocation is salt sensitive, we conclude that the dimeric state of SecA is required for protein translocation. A structural model for the dimeric assembly of SecA while bound to SecYEG is proposed based on the crystal structures of the Thermotoga maritima SecA-SecYEG and the Escherichia coli SecA dimer.
authors.affiliation: Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, and Zernike Institute for Advanced Materials, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
authors.forename: Ilja
authors.initials: I
authors.lastname: Kusters
authors.forename: Geert
authors.initials: G
authors.lastname: van den Bogaart
authors.forename: Alexej
authors.initials: A
authors.lastname: Kedrov
authors.forename: Victor
authors.initials: V
authors.lastname: Krasnikov
authors.forename: Faizah
authors.initials: F
authors.lastname: Fulyani
authors.forename: Bert
authors.initials: B
authors.lastname: Poolman
authors.forename: Arnold J M
authors.initials: AJ
authors.lastname: Driessen
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D011510:Proteolipids / Q000737:chemistry; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D012965:Sodium Chloride; D012996:Solutions; D020124:Thermotoga maritima / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D021122:Protein Subunits; D011510:Proteolipids; D000069816:SEC Translocation Channels; D012996:Solutions; C020485:proteoliposomes; D012965:Sodium Chloride; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins